Michael Skovbo Windahl

Probing substrate interactions in the active tunnel of a catalytically deficient cellobiohydrolase (Cel7)

Background: Substrate interactions in the long tunnel of processive cellulases govern both their catalytic activity and stepwise movement along a cellulose strand. Results: The energetics of enzyme-substrate interactions at different depths of the tunnel are reported. Conclusion: The affinity for the substrate varies strongly through the tunnel. Significance: Quantitative information on interactions is required to understand the complex processive mechanism. Cellobiohydrolases break down cellulose sequentially by sliding along the crystal surface with a single cellulose strand threaded through the catalytic tunnel of the enzyme. This so-called processive mechanism relies on a complex pattern of enzyme-substrate interactions, which need to be addressed in molecular descriptions of processivity and its driving forces. Here, we have used titration calorimetry to study interactions of cellooligosaccharides (COS) and a catalytically deficient variant (E212Q) of the enzyme Cel7A from Trichoderma reesei. This enzyme has ∼10 glucopyranose subsites in the catalytic tunnel, and using COS ligands with a degree of polymerization (DP) from 2 to 8, different regions of the tunnel could be probed. For COS ligands with a DP of 2–3 the binding constants were around 105 m−1, and for longer ligands (DP 5–8) this value was ∼107 m−1. Within each of these groups we did not find increased affinity as the ligands got longer and potentially filled more subsites. On the contrary, we found a small but consistent affinity loss as DP rose from 6 to 8, particularly at the higher investigated temperatures. Other thermodynamic functions (ΔH, ΔS, and ΔCp) decreased monotonously with both temperature and DP. Combined interpretation of these thermodynamic results and previously published structural data allowed assessment of an affinity profile along the length axis of the active tunnel.

Temperature Effects on Kinetic Parameters and Substrate Affinity of Cel7A Cellobiohydrolases

Background: Temperature concomitantly modulates kinetic and adsorption properties in heterogeneous enzyme catalysis. Results: Affinity-activity relationships for four Cel7A cellobiohydrolases are characterized over a broad temperature interval. Conclusion: Cellobiohydrolases are strongly activated by temperature at high, but not at low, substrate loads. Significance: Fundamental insight into cellulolytic mechanisms at high (industrially relevant) temperatures is gained. We measured hydrolytic rates of four purified cellulases in small increments of temperature (10–50 °C) and substrate loads (0–100 g/liter) and analyzed the data by a steady state kinetic model that accounts for the processive mechanism. We used wild type cellobiohydrolases (Cel7A) from mesophilic Hypocrea jecorina and thermophilic Rasamsonia emersonii and two variants of these enzymes designed to elucidate the role of the carbohydrate binding module (CBM). We consistently found that the maximal rate increased strongly with temperature, whereas the affinity for the insoluble substrate decreased, and as a result, the effect of temperature depended strongly on the substrate load. Thus, temperature had little or no effect on the hydrolytic rate in dilute substrate suspensions, whereas strong temperature activation (Q10 values up to 2.6) was observed at saturating substrate loads. The CBM had a dual effect on the activity. On one hand, it diminished the tendency of heat-induced desorption, but on the other hand, it had a pronounced negative effect on the maximal rate, which was 2-fold larger in variants without CBM throughout the investigated temperature range. We conclude that although the CBM is beneficial for affinity it slows down the catalytic process. Cel7A from the thermophilic organism was moderately more activated by temperature than the mesophilic analog. This is in accord with general theories on enzyme temperature adaptation and possibly relevant information for the selection of technical cellulases.

In Situ Stability of Substrate-Associated Cellulases Studied by DSC

This work shows that differential scanning calorimetry (DSC) can be used to monitor the stability of substrate-adsorbed cellulases during long-term hydrolysis of insoluble cellulose. Thermal transitions of adsorbed enzyme were measured regularly in subsets of a progressing hydrolysis, and the size of the transition peak was used as a gauge of the population of native enzyme. Analogous measurements were made for enzymes in pure buffer. Investigations of two cellobiohydrolases, Cel6A and Cel7A, from Trichoderma reesei, which is an anamorph of the fungus Hypocrea jerorina, showed that these enzymes were essentially stable at 25 °C. Thus, over a 53 h experiment, Cel6A lost less than 15% of the native population and Cel7A showed no detectable loss for either the free or substrate-adsorbed state. At higher temperatures we found significant losses in the native populations, and at the highest tested temperature (49 °C) about 80% Cel6A and 35% of Cel7A was lost after 53 h of hydrolysis. The data consistently showed that Cel7A was more long-term stable than Cel6A and that substrate-associated enzyme was less long-term stable than enzyme in pure buffer stored under otherwise equal conditions. There was no correlation between the intrinsic stability, specified by the transition temperature in the DSC, and the long-term stability derived from the peak area. The results are discussed with respect to the role of enzyme denaturation for the ubiquitous slowdown observed in the enzymatic hydrolysis of cellulose.

Reversibility of Substrate Adsorption for the Cellulases Cel7A, Cel6A, and Cel7B from Hypocrea jecorina

Adsorption of cellulases on the cellulose surface is an integral part of the catalytic mechanism, and a detailed description of the adsorption process is therefore required for a fundamental understanding of this industrially important class of enzymes. However, the mode of adsorption has proven intricate, and several key questions remain open. Perhaps most notably it is not clear whether the adsorbed enzyme is in dynamic equilibrium with the free population or irreversibly associated with no or slow dissociation. To address this, we have systematically investigated adsorption reversibility for two cellobiohydrolases (Cel7A and Cel6A) and one endoglucanase (Cel7B) on four types of pure cellulose substrates. Specifically, we monitored dilution-induced release of adsorbed enzyme in samples that had previously been brought to a steady state (constant concentration of free enzyme). In simple dilution experiments (without centrifugation), the results consistently showed full reversibility. In contrast to this, resuspension of enzyme-substrate pellets separated by centrifugation showed extensive irreversibility. We conclude that these enzymes are in a dynamic equilibrium between free and adsorbed states but suggest that changes in the physical properties of cellulose caused by compaction of the pellet hampers subsequent release of adsorbed enzyme. This latter effect may be pertinent to both previous controversies in the literature on adsorption reversibility and the development of enzyme recycling protocols in the biomass industry.

Loop variants of the thermophile Rasamsonia emersonii Cel7A with improved activity against cellulose.

Cel7A cellobiohydrolases perform processive hydrolysis on one strand of cellulose, which is threaded through the enzymes substrate binding tunnel. The tunnel structure results from a groove in the catalytic domain, which is covered by a number of loops. These loops have been identified as potential targets for engineering of this industrially important enzyme family, but only few systematic studies on this have been made. Here we show that two asparagine residues (N194 and N197) positioned in the loop covering the glucopyranose subsite −4 (recently denoted B2 loop) of the thermostable Cel7A from Rasamsonia emersonii had profound effects on both substrate interactions and catalytic efficacy. At room temperature the double mutant N194A/N197A showed strongly reduced substrate affinity with a water‐cellulose partitioning coefficient threefold lower than the wild type. Yet, this variant was catalytically efficient with a maximal turnover about twice as high as the wild type. Analogous but smaller changes were found for the single mutants. Analysis of these changes in affinity and kinetics as a function of temperature, led to the conclusion that replacement of N194 and particularly N197 with alanine leads to faster enzyme‐substrate dissociation. Conversely, these residues appeared to have little or no effect on the rate of association. We suggest that the controlled adjustment of the enzyme‐substrate dissociation prompts faster cellulolytic enzymes. Biotechnol. Bioeng. 2017;114: 53–62.

The influence of different linker modifications on the catalytic activity and cellulose affinity of cellobiohydrolase Cel7A from Hypocrea jecorina

Various cellulases consist of a catalytic domain connected to a carbohydrate-binding module (CBM) by a flexible linker peptide. The linker if often strongly O-glycosylated and typically has a length of 20-50 amino acid residues. Functional roles, other than connecting the two folded domains, of the linker and its glycans, have been widely discussed, but experimental evidence remains sparse. One of the most studied cellulose degrading enzymes is the multi-domain cellobiohydrolase Cel7A from Hypocrea jecorina. Here, we designed variants of Cel7A with mutations in the linker region to elucidate the role of the linker. We found that moderate modification of the linker could result in significant changes in substrate affinity and catalytic efficacy. These changes were quite different for different linker variants. Thus, deletion of six residues near the catalytic domain had essentially no effects on enzyme function. Conversely, a substitution of four glycosylation sites near the middle of the linker reduced substrate affinity and increased maximal turnover. The observation of weaker binding provides some support of recent suggestions that linker glycans may be directly involved in substrate interactions. However, a variant with several inserted glycosylation sites near the CBM also showed lower affinity for the substrate compared to the wild-type, and we suggest that substrate interactions of the glycans depend on their exact location as well as other factors such as changes in structure and dynamics of the linker peptide.

Exo-exo synergy between Cel6A and Cel7A from Hypocrea jecorina: Role of carbohydrate binding module and the endo-lytic character of the enzymes

Synergy between cellulolytic enzymes is essential in both natural and industrial breakdown of biomass. In addition to synergy between endo‐ and exo‐lytic enzymes, a lesser known but equally conspicuous synergy occurs among exo‐acting, processive cellobiohydrolases (CBHs) such as Cel7A and Cel6A from Hypocrea jecorina. We studied this system using microcrystalline cellulose as substrate and found a degree of synergy between 1.3 and 2.2 depending on the experimental conditions. Synergy between enzyme variants without the carbohydrate binding module (CBM) and its linker was strongly reduced compared to the wild types. One plausible interpretation of this is that exo‐exo synergy depends on the targeting role of the CBM. Many earlier works have proposed that exo‐exo synergy was caused by an auxiliary endo‐lytic activity of Cel6A. However, biochemical data from different assays suggested that the endo‐lytic activity of both Cel6A and Cel7A were 103–104 times lower than the common endoglucanase, Cel7B, from the same organism. Moreover, the endo‐lytic activity of Cel7A was 2–3‐fold higher than for Cel6A, and we suggest that endo‐like activity of Cel6A cannot be the main cause for the observed synergy. Rather, we suggest the exo‐exo synergy found here depends on different specificities of the enzymes possibly governed by their CBMs. Biotechnol. Bioeng. 2017;114: 1639–1647.